Alternative splicing is a powerful regulatory mechanism for generating subtle changes in isotype distribution. The insulin receptor itself is alternatively spliced in a developmental and tissue-specific manner. A change in distribution of the two isotypes has the potential to contribute to insulin resistance since the two isotypes possess different insulin responsive properties. Differing reports on changes in the insulin receptor isotype in diabetic and control subjects prompted us to examine the isotype distribution in Pima subjects and to characterize a potential role for insulin itself in the regulation of the alternative splicing of the insulin receptor. A small and significant difference in insulin receptor isotype was detected in insulin resistant Pima subjects compared to insulin sensitive Pima subjects using RT-PCR of RNA prepared from skeletal muscle biopsies. Insulin transiently influenced the insulin receptor isotype distribution following insulin treatment of FAO cells, a rat insulin-sensitive hepatoma cell line. These insulin inducible changes in insulin receptor isotype distribution are thought to occur as a consequence of hyperinsulinemic response to insulin resistance which has some other cause(s). Changes in the isotype distribution may be a useful biochemical marker for identifying subclinically insulin resistant subjects at an increased risk for developing diabetes. We plan to compare insulin receptor isotype distributions in individuals, who are insulin sensitive but have a family history of diabetes, to determine if changes in isotype distribution are present prior to the presence of clinically detectable changes in insulin-sensitivity. These findings will be expanded to include other putative insulin responsive messenger RNAs as candidates for marker development (T. Cooper). Fractionated blood cells will be used to carry out these experiments since these cells can be easily obtained and display sensitivity toward insulin.